Rapamycin is the only compound that has been unambiguously shown to extend the maximum lifespan of mice. Unfortunately, side effects including immunosuppression and the elevation of cardiovascular risk factors are likely to limit the utility of the drug in humans. Therefore, there is a great need and opportunity to understand how rapamycin works - both for the development of safe and effective therapeutics, and to gain insight into the basic mechanisms of aging itself. The canonical target of rapamycin is mTORC1, a nutrient sensing kinase whose homolog has been implicated in the extension of lifespan by caloric restriction (CR) in lower organisms. In mice, ablation of the mTORC1 target S6 kinase 1 (S6K1) mimics salient features of CR, including increases in insulin sensitivity, mitochondrial biogenesis, and lifespan. Therefore, it has been postulated that rapamycin mimics CR by inhibiting the mTORC1/S6K1 axis in mammals. In sharp contrast to CR, however, rapamycin actually causes insulin resistance and, at least in cells, inhibits oth the production and activity of mitochondria. These are surprising and potentially very important observations, given that both insulin sensitization and increased mitochondrial biogenesis have been suggested to contribute to CR-induced longevity. We recently showed that rapamycin-induced insulin resistance is the result of inhibiting a second target, mTORC2, and moreover, that specific inhibition of mTORC1 extends lifespan without detrimental effects on insulin signaling. Next, we plan to test whether the inhibition of mitochondrial biogenesi and activity that is observed in cells also occurs in vivo. If so, rapamycin will allow us to proide the first clear demonstration that mitochondrial biogenesis can be uncoupled from longevity. In a second line of experiments, we will treat S6K1 knockout mice with rapamycin to test the hypothesis that S6K1-independent mechanisms contribute to its effects on longevity. There are a number of reasons for believing that this will be the case. S6K1 ablation produces very different changes in physiology and does not extend life in males, whereas rapamycin does. Moreover, the mTORC2 homolog regulates longevity in worms, and our demonstration that rapamycin disrupts mTORC2 in mice therefore provides a candidate mechanism for S6K1-independent effects. Finally, we will explore the tissue-specific consequences of mTORC2 disruption. Loss of mTORC2 in the liver appears to mediate detrimental effects of rapamycin on insulin sensitivity, and ameliorating these effects could lead to complementary approaches to improve the safety and efficacy of the drug. On the other hand, loss of another insulin signaling molecule, IRS2, in the brain has previously been shown to extend life, and loss of neuronal mTORC2 might therefore contribute to the beneficial effect of rapamycin on lifespan. Elucidating the mechanisms by which rapamycin is able to prevent or slow progression of age-related diseases and extend the maximum survival time in mice will offer important insights, and likely new therapeutic targets, in the effort to promote healthy human aging.